SYBR green: melt curve Tm shift

Ed Siefker via methods%40net.bio.net (by ebs15242 from creighton.edu)
Mon Dec 21 10:57:39 EST 2009


I am having a small problem with my SYBR green qrtpcr.
I have 4 primer sets running against 2 different cDNA
samples.  The primers are all at the same concentration
(300 nm). I prepared both cDNA samples side by side, in
exactly the same way and loaded 10ng of each.

The problem is, when I look at the melt curve, I'm seeing
a decrease in Tm of the product of about 1 degree C from
one cDNA to another.  This Tm shift occurs for all 4 primer
sets. In both cases, there is a single  sharp peak, so I'm
doubting I have primer dimers.

Does anyone have any ideas?  I've used these primers before
on different samples, and the melting curves always exactly
coincided, no matter what the cDNA was.



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